2.1 MDON and their Use to Effect Specific Genetic Alterations
Mixed duplex oligonucleotides (MDON) and their use to effect genetic changes in eukaryotic cells are described in U.S. Pat. No. 5,565,350 to Kmiec (Kmiec I). Kmiec I discloses inter alia MDON having two strands, in which a first strand contains two segments of at least 8 RNA-like nucleotides that are separated by a third segment of from 4 to about 50 DNA-like nucleotides, termed an “interposed DNA segment.” The nucleotides of the first strand are base paired to DNA-like nucleotides of a second strand. The first and second strands are additionally linked by a segment of single stranded nucleotides so that the first and second strands are parts of a single oligonucleotide chain. Kmiec I further teaches a method for introducing specific genetic alterations into a target gene. According to Kmiec I, the sequences of the RNA segments are selected to be homologous, i.e., identical, to the sequence of a first and a second fragment of the target gene. The sequence of the interposed DNA segment is homologous with the sequence of the target gene between the first and second fragment except for a region of difference, termed the “heterologous region.” The heterologous region can effect an insertion or deletion, or can contain one or more bases that are mismatched with the sequence of target gene so as to effect a substitution. According to Kmiec I, the sequence of the target gene is altered as directed by the heterologous region, such that the target gene becomes homologous with the sequence of the MDON. Kmiec I specifically teaches that ribose and 2′-Omethylribose, i.e., 2′-methoxyribose, containing nucleotides can be used in MDON and that naturally-occurring deoxyribose-containing nucleotides can be used as DNA-like nucleotides.
U.S. patent application Ser. No. 08\664,487, filed Jun. 17, 1996, now U.S. Pat. No. 5,731,181 (Kmiec II) does specifically disclose the use of MDON to effect genetic changes in plant cells and discloses further examples of analogs and derivatives of RNA-like and DNA-like nucleotides that can be used to effect genetic changes in specific target genes.
Scientific publications disclosing uses of MDON having interposed DNA segments include Yoon, et al., 1996, Proc. Natl. Acad. Sci. 93:2071–2076 and Cole-Straus, A. et al., 1996, SCIENCE 273:1386–1389. The scientific publications disclose that rates of mutation as high as about one cell in ten can be obtained using liposomal mediated delivery. However, the scientific publications do not disclose that MDON can be used to make genetic changes in plant cells.
The present specification uses the term MDON, which should be understood to be synonymous with the terms “chimeric mutation vector,” “chimeric repair vector” and “chimeraplast” which are used elsewhere.
2.2 Transgenic Plant Cells and the Generation of Plants from Transgenic Plant Cells
Of the techniques taught by Kmiec I and II for delivery of MDON into the target cell, the technique that is most applicable for use with plant cells is the electroporation of protoplasts. The regeneration of fertile plants from protoplast cultures has been reported for certain species of dicotyledonous plants, e.g., Nicotiana tobacum (tobacco), U.S. Pat. No. 5,231,019 and Fromm, M. E., et al., 1988, Nature 312, 791, and soybean variety Glycine max, WO 92/17598 to Widholm, J. M. However, despite the reports of isolated successes using non-transformed cells, Prioli, L. M., et al., Bio/technology 7, 589, Shillito, R. D., et al., 1989, Bio/Technology 7, 581, the regeneration of fertile monocotyledonous plants from transformed protoplast cultures is not regarded as obtainable with application of routine skill. Frequently, transformed protoplasts of monocotyledonous plants result in non-regenerable tissue or, if the tissue is regenerated the resultant plant is not fertile.
Other techniques to obtain transformed plant cells by introducing kilobase-sized plasmid DNA into plant cells having intact or partially intact cell walls have been developed. U.S. Pat. No. 4,945,050, U.S. Pat. No. 5,100,792 and U.S. Pat. No. 5,204,253 concern the delivery of plasmids into intact plant cells by adhering the plasmid to a microparticle that is ballistically propelled across the cell wall, hereafter “biolistically transformed” cell. For example U.S. Pat. No. 5,489,520 describes the regeneration of a fertile maize plant from a biolistically transformed cell. Other techniques for the introduction of plasmid DNA into suspensions of plant cells having intact cell walls include the use of silicon carbide fibers to pierce the cell wall, see U.S. Pat. No. 5,302,523 to Coffee R., and Dunwell, J. M.
A technique that allows for the electroporation of maize cells having a complex cell wall is reported in U.S. Pat. No. 5,384,253 to Krzyzek, Laursen and P. C. Anderson. The technique uses a combination of the enzymes endopectin lyase (E.C. 3.2.1.15) and endopolygalacturonase (E.C. 4.2.2.3) to generate transformation competent cells that can be more readily regenerated into fertile plants than true protoplasts. However, the technique is reported to be useful only for F1 cell lines from the cross of line A188×line B73.